Sigrid Van Dyck

Michaelis-Menten analysis of bovine plasma amine oxidase by capillary electrophoresis using electrophoretically mediated microanalysis in a partially filled capillary.

A method for determining bovine plasma amine oxidase (PAO; EC 1.4.3.6) activity with benzylamine (Bz) as substrate is described. Electrophoretically mediated microanalysis (EMMA) combined with micellar electrokinetic capillary chromatography (MEKC) was used to perform an on‐capillary enzymatic reaction and to separate the generated benzaldehyde from the other reaction products. The capillary was only partially filled with the separation solution, since the enzyme was unstable in the presence of the applied surfactant. The initial reaction velocity of the enzyme‐catalyzed reaction was estimated from the peak area of the enzyme product, benzaldehyde. An amplification step was introduced by means of an on‐capillary incubation of 15 min, in order to accumulate enough reaction product to detect spectrophotometrically at 254 nm. This set‐up resulted in a fully automated assay, which can be carried out in less then 35 min. Using the Lineweaver‐Burk equation, an average Michaelis constant (KM) for PAO was calculated to be 0.74 mM ± 0.05 mM, which is consistent with previously reported values.

Kinetic study of angiotensin converting enzyme activity by capillary electrophoresis after in-line reaction at the capillary inlet

The in-capillary reaction of angiotensin converting enzyme (ACE) with the tripeptide substrate hippuryl-L-histidyl-L-leucine was studied. ACE activity was determined by the quantitation of the product, hippuric acid, at 230 nm. Reaction occurred at the capillary inlet during a predetermined waiting period, followed by the electrophoretic separation of the compounds. When the set-up was reversed, i.e. reaction at the opposite side after short-end injection of enzyme and substrate, separation was achieved in less than 5 min. Using the Lineweaver-Burk equation, an average Michaelis constant for ACE from rabbit lung was calculated to be 1.16 +/- 0.12 mM, a value consistent with previously reported data.

Kinetic study of γ-glutamyltransferase activity by electrophoretically mediated microanalysis combined with micellar electrokinetic capillary chromatography

The use of capillary electrophoresis for the determination of γ‐glutamyltransferase (GGT) activity with γ‐glutamyl‐p‐nitroanilide (Glu‐p‐NA) as a substrate was investigated. The reaction velocity was quantified spectrophotometrically by the corrected peak area of the product p‐nitroaniline (pNA) at 380 nm. Micelles composed of sodium deoxycholic acid were used in the background electrolyte in order to obtain a baseline separation between the substrate and the product. The presence of the micelles did not influence the enzymatic reaction. The electrophoretic system was used, not only for the separation and quantitation of the different reaction compounds but also for the in‐capillary mixing of the enzyme and substrate plugs. This methodology is known as electrophoretically mediated microanalysis (EMMA). With the developed in‐capillary activity assay an average Michaelis constant (KM) for GGT was calculated to be 2.09 mM (RSD = 7.3%, n = 3), a value consistent with previously reported values.

Inhibition study of angiotensin converting enzyme by capillary electrophoresis after enzymatic reaction at capillary inlet.

Capillary electrophoresis was used to study the inhibition of angiotensin-converting enzyme (ACE) by different inhibitors. Reaction occurred at the capillary inlet during a predetermined waiting period, followed by the electrophoretic separation of the reaction compounds. ACE activity was determined by the quantification of the reaction product, hippuric acid, at 230 nm. The technique was used to study the potency of five different inhibitors (captopril, lisinopril, perindoprilat, quinaprilat and benazeprilat). During a kinetic study, the Ki value of captopril was estimated to be 55.4 +/- 8.8 nM, a value consistent with previously reported values.